The use of automated quantitative analysis to evaluate epithelial-to-mesenchymal transition associated proteins in clear cell renal cell carcinoma.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) has recently been implicated in the initiation and progression of renal cell carcinoma (RCC). Some mRNA gene expression studies have suggested a link between the EMT phenotype and poorer clinical outcome from RCC. This study evaluated expression of EMT-associated proteins in RCC using in situ automated quantitative analysis immunofluorescence (AQUA) and compared expression levels with clinical outcome. METHODS/PRINCIPAL FINDINGS: Unsupervised hierarchical cluster analysis of pre-existing RCC gene expression array data (GSE16449) from 36 patients revealed the presence of an EMT transcriptional signature in RCC [E-cadherin high/SLUG low/SNAIL low]. As automated immunofluorescence technology is dependent on accurate definition of the tumour cells in which measurements take place is critical, extensive optimisation was carried out resulting in a novel pan-cadherin based tumour mask that distinguishes renal cancer cells from stromal components. 61 patients with ccRCC and clinical follow-up were subsequently assessed for expression of EMT-associated proteins (WT1, SNAIL, SLUG, E-cadherin and phospho-β-catenin) on tissue microarrays. Using Kaplan-Meier analysis both SLUG (p = 0.029) and SNAIL (p = 0.024) (log rank Mantel-Cox) were significantly associated with prolonged progression free survival (PFS). Using Cox regression univariate and multivariate analysis none of the biomarkers were significantly correlated with outcome. 14 of the 61 patients expressed the gene expression analysis predicted EMT-protein signature [E-cadherin high/SLUG low/SNAIL low], which was not found to be associated to PFS when measured at the protein level. A combination of high expression of SNAIL and low stage was able to stratify patients with greater significance (p = 0.001) then either variable alone (high SNAIL p = 0.024, low stage p = 0.029). CONCLUSIONS: AQUA has been shown to have the potential to identify EMT related protein targets in RCC allowing for stratification of patients into high and low risk groups, as well the ability to assess the association of reputed EMT signatures to progression of the disease

The use of automated quantitative analysis to evaluate epithelial-to-mesenchymal transition associated proteins in clear cell renal cell carcinoma.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) has recently been implicated in the initiation and progression of renal cell carcinoma (RCC). Some mRNA gene expression studies have suggested a link between the EMT phenotype and poorer clinical outcome from RCC. This study evaluated expression of EMT-associated proteins in RCC using in situ automated quantitative analysis immunofluorescence (AQUA) and compared expression levels with clinical outcome. METHODS/PRINCIPAL FINDINGS: Unsupervised hierarchical cluster analysis of pre-existing RCC gene expression array data (GSE16449) from 36 patients revealed the presence of an EMT transcriptional signature in RCC [E-cadherin high/SLUG low/SNAIL low]. As automated immunofluorescence technology is dependent on accurate definition of the tumour cells in which measurements take place is critical, extensive optimisation was carried out resulting in a novel pan-cadherin based tumour mask that distinguishes renal cancer cells from stromal components. 61 patients with ccRCC and clinical follow-up were subsequently assessed for expression of EMT-associated proteins (WT1, SNAIL, SLUG, E-cadherin and phospho-β-catenin) on tissue microarrays. Using Kaplan-Meier analysis both SLUG (p = 0.029) and SNAIL (p = 0.024) (log rank Mantel-Cox) were significantly associated with prolonged progression free survival (PFS). Using Cox regression univariate and multivariate analysis none of the biomarkers were significantly correlated with outcome. 14 of the 61 patients expressed the gene expression analysis predicted EMT-protein signature [E-cadherin high/SLUG low/SNAIL low], which was not found to be associated to PFS when measured at the protein level. A combination of high expression of SNAIL and low stage was able to stratify patients with greater significance (p = 0.001) then either variable alone (high SNAIL p = 0.024, low stage p = 0.029). CONCLUSIONS: AQUA has been shown to have the potential to identify EMT related protein targets in RCC allowing for stratification of patients into high and low risk groups, as well the ability to assess the association of reputed EMT signatures to progression of the disease

Multivariate Cox Regression Analysis of clinicopathological variables and EMT biomarkers against PFS.

<p>*Significant at p<0.05 level.</p

Progression free survival (PFS) curves based on combination of low stage and high SNAIL protein expression.

<p>Note that 13 patients were classed as having 0 months PFS due to metastatic disease at the time of nephrectomy.</p

Clinicopathological characteristics of ccRCC patients included in study TMA (n = 61).

<p>Clinicopathological characteristics of ccRCC patients included in study TMA (n = 61).</p

AQUA quantitative image analysis.

<p>DAPI counterstain (blue) was used to identify nuclei, pan-cadherin antibody (rabbit) or a combination of pan-cadherin and pan-cytokeratin antibodies (mouse) to identify infiltrating tumour cells (green) and Cy-5-tyramide for detection of following target proteins (red) (a. E-cadherin, b. SNAIL, c. SLUG, d.WT1 and e. phospho-β-catenin).</p

Survival curves based on AQUA protein expression cut-off points.

<p>(a) E-cadherin expression, (b) SNAIL expression, (c) SLUG expression and (d) EMT phenotype versus progression free survival (PFS). Note that 13 patients were classed as having 0 months PFS due to metastatic disease at the time of nephrectomy.</p

Progression free survival (PFS) curves.

<p>(a) stage and (b) grade using Kaplan-Meier methods. Note that 13 patients were classed as having 0 months PFS due to having metastatic disease at the time of nephrectomy.</p

Unsupervised hierarchical clustering of the GSE16449 microarray dataset, using the probe-sets of interest (Table S1).

<p>Cluster 1 represents the group expressing the EMT signature and increased Wnt signalling, whereas the Cluster 2 illustrates the non-EMT cluster group. (green = low expression, red = high expression).</p

Images of selected tumour masking options.

<p>(a) Vimentin (stroma stained) (b) Renal cell marker (stroma and epithelia stained) (c) Pan-cadherin and CK5/6/8/18 (representative tumour mask for optimised mouse tumour mask) (d) Pan-cadherin (representative tumour mask for rabbit tumour mask). White arrows indicate areas of non specific binding.</p